Previous studies manifested that microRNA-145-5p is pivotal in the development of various cancers. Nevertheless, the potential function of microRNA-145-5p in colorectal cancer remains unclear. This study attempted to investigate the potential role and possible mechanism of microRNA-145-5p in colon cancer. MicroRNA-145-5p and phosphoserine aminotransferase 1 (PSAT1) levels in colon cancer cells were assayed via quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell proliferation and cell cycle status were assessed using Cell Counting Kit-8, colony formation, and flow cytometry. The target binding relationship of microRNA-145-5p and PSAT1 was identified using bioinformatics analysis and dual-luciferase reporter gene assay. The result of qRT-PCR disclosed that microRNA-145-5p was markedly down-regulated and PSAT1 level was up-regulated in colon cancer cell lines. Besides, enforced microRNA-145-5p level repressed proliferation of colon cancer cells, and cells were arrested in G0-G1 phase. Bioinformatics analysis and dual-luciferase reporter genes confirmed that PSAT1 was a downstream target of microRNA-145-5p. Enforced PSAT1 level remarkably modulated cell cycle and fostered cell proliferation. Furthermore, rescue experiments displayed that microRNA-145-5p restrained cell cycle progression and cell proliferation and forced PSAT1 level could partially reverse this process. Taken together, our findings demonstrated that microRNA-145-5p repressed colon cancer cell cycle progression and cell proliferation via targeting PSAT1. Our findings identified microRNA-145-5p as an essential tumor repressor gene in colon cancer and may provide a novel biomarker for colon cancer. Graphical abstract